International Journal of Aquaculture, 2017, Vol.7, No.10, 71-78
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2 Materials and Methods
2.1 Experimental feed
Commercial pellet feed (CP 9931, Charoen Pokphand Pvt. Ltd., India) containing 30% protein, 3% lipid, 8% fibre,
12% of moisture, and 47% ash and nitrogen-free extract was used.
2.2 Experimental fish and design
2.2.1 Captive rearing of IMCs
Apparently healthy sub-adult IMCs, viz.,
Labeo rohita
(348.50±9.53 g),
Catla catla
(350.89±8.53 g) and
Cirrhinus mrigala
(345.25±9.93 g) were collected from a commercial grow-out pond in Naihati (Lat. 22°54’04” N;
Long. 88°25’38’’ E), North 24 Parganas district, West Bengal, India. The fish, on receipt, were disinfected with 5
ppm potassium permanganate solution and conditioned for 21 days in circular fibreglass reinforced plastic (FRP)
tanks of 500 L capacity containing 300 L bore-well water with continuous aeration. Manual water exchange
(20-25%) was done on alternate days after syphoning out the uneaten feed and excreta. Each of the three species
was stocked separately at 15 fish/tank, in triplicate and fed with commercial pellet feed at 3% of the body weight
twice daily.
2.2.2 Culture of IMCs in net cages in normal and sewage-fed ponds
Experiments were simultaneously conducted in a normal pond of size 0.33 ha situated at Chakgaria, Kolkata,
India (Lat. 22°82’ N; Long. 88°20’ E) and also in a sewage-fed pond (locally called
bhery
) of size 18.67 ha
situated at Haripota, South 24 Parganas district, West Bengal, India (Lat. 22°29’ N; Long. 88°29’ E). Apparently
healthy juvenile to sub-adult
C. catla
(150.61±16.38 g),
L. rohita
(101.40±16.60 g) and
C. mrigala
(121.64±24.82
g) for rearing in normal pond were procured from Gangajoara (Lat. 22°27’ N; Long. 88°26’ E), South 24 Parganas
district, transported in double layer oxygen packed polyethylene bags and acclimatised as above. Likewise,
healthy juvenile to sub-adult
C. catla
(148.61±12.38 g),
L. rohita
(111.80±14.60 g) and
C. mrigala
(132.34
±
18.92 g) for rearing in the sewage-fed pond were harvested from the same farm complex. The details of culture
and management practices followed in normal and sewage-fed farms are presented in Table 1.
The experiments were conducted in a 2 (farm) ×3 (species) factorial design in triplicate. Six numbers of net cages,
each with the dimension of 2.43 m ×1.37 m ×1.37 m, were erected by the support of bamboo poles in each pond.
The net cage is a fixed and fine-meshed net enclosure, similar to an inverted mosquito net made out of nylon
netting with joints in nylon thread, double stitched to prevent splitting. Individual net cage was fixed in such a
way that one-third portion of the net cage remained above the water surface to allow the surfacing of fish. Healthy
and active
C. catla
,
L. rohita
and
C. mrigala
were stocked in each net cage @ 18 fish/cage, 6 individuals of each
species in the ratio 1:1:1. An additional cage was also maintained with few individuals of each species in both
ponds for the emergency requirement. Fish were conditioned for 21 days in the net cage. During the period of
acclimatisation, the dead ones were replaced immediately with respective fish species of the uniform size. The
fish were fed with pellet feed at 3% of the body weight twice daily.
2.3 Determination of water quality parameters
The temperature of the water was recorded by centigrade thermometer at the site. The pH of water samples was
estimated by pH meter (Eutech Instruments Pte Ltd). Total dissolved solids (TDS) were measured by TDS meter
(HiMedia, India) and expressed as mg/L. The biological oxygen demand (BOD), dissolved oxygen, free carbon
dioxide, phosphate-phosphorous, ammonia-nitrogen and total hardness were determined by APHA/AWWA/WEF
(2005) methods and expressed as mg/L (Table 2).
2.4 Blood collection and determination of serum insulin-like growth factor- 1 (IGF-1)
The sterile disposable syringe of 2 ml capacity with 23G needle (Hindustan Syringes and Medical Devices Ltd,
India) was used for each fish for the collection of blood. On day 21, the blood (min: 0.5 ml and max: 1.5 ml/fish)
was collected by the caudal venous puncture from five fish of each replicate for each species of the normal pond,
sewage-fed pond and FRP tanks. The blood was allowed to clot overnight at 4ºC by keeping the syringes at 15º
angle. Serum from the syringes was then carefully poured-out into Eppendorf tubes and centrifuged in a
